This research focuses on oxidative modification of proteins. The resulting covalent modifications have been implicated in important physiologic and pathologic processes. Determination of the actual roles of oxidative modification in these processes requires the identification of specific proteins that are susceptible to modification and the mapping of the sites of modification in those proteins. During this year, we placed increased emphasis on the in vivo detection of oxidatively modified proteins. The modifications being examined include oxidation of methionine to methionine sulfoxide, of arginine and lysine to their semi-aldehydes, and of cysteine to the disulfide with glutathione. Analytical methodology was developed to facilitate quantitative analysis for site-specific oxidations of these residues. For example, in collaboration with the Pulmonary Branch of NHLBI, we quantitatively assessed the oxidation of both methionine-351 and methionine-358 in alpha-1-antitrypsin of lung lavage from volunteers who are either non-smokers or smokers. Having identified proteins that are sensitive to various oxidative modifications, we can now study the physiologic and pathologic effects of the modifications and consider rational intervention to modulate the extent of modification. Taking the alpha-1-antitrypsin example again, we showed that the biological activity of this protease inhibitor can be stabilized against oxidative inactivation by converting the two methionine residues to valine, thus preventing the introduction of methionine sulfoxide into the protein.